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Int. J. LifeSc. Bt & Pharm. Res. 2014 Sushen Pradhan et al., 2014

IN VITRO MICROPROPAGATION OF AMOMUM

SUBULATUM (ZINGIBERACEAE), A MAJORTRADITIONAL CASH CROP OF SIKKIM HIMALAYA

Sushen Pradhan1*, Smrita Pradhan1, Bharat C Basistha1 and K B Subba1

Research Paper

Present investigation was achieved with an efficient, simple micropropagation methods wasstandardized for Ammomum subalatum cv. Ramsey for the production of disease free andrapid multiplication of planting material of Large cardomom micro-propagules and conservationof genetic resources of large cultivator variety of Sikkim. Modified MS medium with 4 % of sucrosewith different concentration of hormone showed the suitable medium for multiplication of largecardamom. A total of 52 different hormone concentrations of growth hormone/ cultured mediumwere examined. The maximum number of shoots and roots were observed on (MS+ sucrose40 g + BAP 3mg/l+0.5 NAA+2mg/l) and second was (MS+ 40g sucrose + BAP 3.5mg/l+0.5 NAA+ 2mg/l). After individually subcultured on same fresh medium, adventitious buds were initiatedafter 10–15 days of culture, complete plantlets developed within 50 days and maintained for 18months without decreasing the multiplication rate. The combination of cytokinins and auxins inthe culture medium enhanced the response in a number of plants in terms of shoot and rootgrowths, with 100% in vitro multipled plantlets were successfully transferred into field.

Keywords: Ammomum subalatum, Micropropagation, Zingibereaceae, Ramsey, Sikkim

*Corresponding Author: Sushen Pradhan � [email protected]

ISSN 2250-3137 www.ijlbpr.com

Vol. 3, No. 2, April 2014

© 2014 IJLBPR. All Rights Reserved

Int. J. LifeSc. Bt & Pharm. Res. 2014

1 Sikkim State Council of Science and Technology, Sajong, Rumtek, East Sikkim.

INTRODUCTION

The genus Amomum subulatum Roxburgh,

(Family: Zingiberaceae, 2n=24 chromosome),

commonly known as”Bada Elaichi” or “Black

Cardamom” is a perennial herb, monocieous,

and monocotyledon (Sajini et al, 1997). However,

variability is also reported with 2n = 26, 34, 42

and 44 (Sharma and Bhattacharya, 1959). It is

one of the oldest spices from Ayurvedic and Unani

preparations dates back to 6th century BC as

mentioned in Sashruta (Sharma, 2000). It was

known to Greeks and Romans as Amomum

subulatum during the 4th century BC and was

recorded by Theophrastus the Greek philosopher

(Mukherjee, 1972).

Roxburgh (1820) was first to illustrated this

plant in his ‘Plants of the Coast of Coromandel’

and in ‘Flora Indica’ (1820). A difference in three

cultivars of A. subulatum (cv-Ramsey, cv-Golsey

and cv-Sawney) was studied by Gyatso et al,

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(1980), Subba (1984) and Rao et al, (1993) of

Sikkim Himalaya. Further, a total of seven cultivar

varieties were distributed in worldwide namely;

cv-Sawney, cv-Seremna, cv-Green Golsey,

cv-Varlangey, cv-Dzongu and cv-Ramsey

(Thomas et al, 2009; Sharma, 2000).

Globally it is cultivated in India, Nepal, China

and Bhutan, and within India Sub-Himalayas

regions like Sikkim, Uttarakhand, Arunachal

Pradesh and West Bengal (Darjeeling and

Kalimpong) states mostly preferred to cultivate

large cardamom (Sharma, 2000).

A.subalatum is a shade loving plant and

requires high moisture areas where annual mean

rain fall varies between 150-350 cm and grows

well under the shade of Alnus nepalensis, Ficus

roxburgii trees in an altitude ranging from 1000 to

2000 m above msl (Anonymous 2003; Sharma

et al, 2010). Since large cardamom cultivation

requires tree shades of its farming, which

indirectly supports conservation of tree

biodiversity. Given these agro climatic

requirements, the state of Sikkim is best suited

for its cultivation. As the crop grows throughout

the year, its temperature requirement varies from

6º C in winter to 30º C in summer (Kashyapi,

2004). About 17000 ha large cardamom holdings

have been recorded in Sikkim (Sharma et al,

2008). In India large cardamom annual production

is around 4000 MT (metric tonne) out of which

Sikkim’s contribute over 85% (Mande et al 1999).

Mostly it is grown on such a slope, which is not

suitable for any crops, and protects the soil from

erosion and landslide (Sharma, 2000). About

17000 ha Cardamom holdings have been

recorded in Sikkim (Sharma et al, 2008).

Large cardamom seed has a pleasant

aromatic odour, due to which it is extensively used

for flavouring vegetables and many food

preparations in India (Pathak, 2008). It also shows

analgesic, antimicrobial, antioxidant and antiulcer

activity (Bisht et al, 2011). The seed contains 2 to

3% of essential oil containing 70 % of 1, 8-cineole

and possesses medicinal properties like

carminative, stomachic, diuretic, cardiac

stimulant, antiemetic and used as flavouring spice

for cooking various culinary dishes, confectionary

and beverage drink (Rao et al, 1993).

Large cardamom is native to the Sikkim Himalaya

(Sharma et al, 2002). The aboriginal inhabitants

of Sikkim have been practicing agroforestry

practices for cultivation of this plant for ages too

boosts their economy leads to sustainability.

Large cardamom agroforestry system not only

provides high income from the cash crops, but it

is also preserve tree biodiversity and obtain

additional income from timber and fuel wood. In

comparison to the other traditional cash crops,

the income from large cardamom has three to

four times higher (Chettri et al, 2013).

Sikkim was the largest producer of large

cardamom in India contributing more than 50 %

share (Bisht et al, 2011) along with Nepal and

Bhutan. Due to its various use, its demand has

been increasing exponentially within country itself

and it is also exported to middle east countries,

Europe, North America and Mainland China

(Madhusoodanan, et al, 2001). However the

situation of the crop over the last few years has

not been encouraging. Decrease in annual

production (from 6,500 mt to 2745 mt) due to

natural calamities and widespread occurrence of

viral and fungal diseases (Sharma et al, 2009).

The productions of large cardamom are

decreasing rapidly due to viral (Foorkey) and

fungal diseases (Chirkey) (Pathak, 2008; Saju et

al, 2013).

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Sikkim farmers are not being able to prevent

their farm from this fatal unidentified disease. On

the other hand, the use of pesticide has not gained

wide acceptance among the farmers who still hold

great faith in their traditional techniques of farming.

They opine that the pesticide would pollute their

water resources and is harmful for their animals

who feed from the same land. The condition is

so severe that the growers who were contributing

approximately three hundred kilograms of fruit in

the market before 4-5 years have nothing to sell

this year. So as a final solution, the farmers are

presently planning to burn the entire farm, keep it

barren for some years and do replantation.

Large cardamom farmers of Sikkim are in

doldrums stage about cultivation of large

cardamom because traditional methods of

propagation with sucker multiplication has some

flaws has led to spread of fungal and viral disease

causing mass reduction of economically

important plants. Seed propagation is also not

preferred by cardamom farmers because of

varations in plant which takes long time for fruiting

(Subba, 1984 Chapter 33). Other reasons for

decline in production of large cardamom were

methods of agricultural practices, lack of quality

planting material, socio-economic conditions, lack

of phytosanitation, and absence of proper shade

management, lack of irrigation facilities and lack

of scientific methods of cultivation (Bhattarai et

al, 2013) anthropogenic activities like

deforestation, forest fire (Sharma et al, 2010).

In this regards, in vitro micro propagation

technique can be a great alternative method for

propagation of large cardamom to farmers. It is

well known that plant tissue culture techniques

have been useful in conservation of germplasms

for vegetatively propagated crops and considered

as an alternative to conventional field gene bank

to safeguard against pests and environmental

vagaries (Dodds, 1991). Three-fold increase in

the production of Zingiberaceae is reported with

effective disease control (Hosoki and Sagawa,

1977; Nadgauda et al, 1980; Pillai and Kumar,

1982; Inden et al, 1988; Bhagyalakshmi and Singh,

1988; Noguchi and Yamakawa, 1988).

Present investigation was carried out for the

standardisation of appropriate protocol for the

production of rapid clonal propagation of

Amomum subulatum Roxb. cv Ramsey for large

scale production and conservation of elite

germplasm. In situ conservation of germplasm

has been considered one of the effective

conservation strategies for conservation

management and preservation of horticulturally

and economically important cash crops in the

world.

MATERIALS AND METHODS

Collection of Samples

Young sucker sample of cv Ramsey was

collected from different cardamom fields of Sikkim

and brought to the laboratory of Sikkim State

Council of Science and Technology, Sajong,

Rumtek for raising in nursery.

Preparation of Explants

Explants for tissue culture of the cultivars of

Ammomum subalatum Roxb. cv Ramsey was

collected from the nursery bed raised in the

Sikkim State Council of Science and Technology,

Rumtek. For preparation of explants standard

sterilization protocol was followed of (Pradhan et

al 2012).

Culture Medium

The different explants (shoot tip and leaf) were

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inoculated in several basal medium namely B5

(Gamborg, 1976), half and full strength of MS

(Murashige and Skoog, 1962) and SH (Schenk

and Hildebrandt, 1972). These different

concentrations of culture media were carried out

for the standardization of best medium for

multiplication and production of disease free

planting materials of Ammomum subalatum

Roxb. cv Ramsey. The growth initiation was

recorded only in shoot tip explants and no growth

was seen for leaf explants. In case of media used,

the growth response were only seen in the MS

(Murashige and Skoog, 1962) media containing

4 % sucrose as the carbon source and no

response were seen in the other medium used.

The growth of the plant in MS medium was

stunned growth and yellowing of leaves.

Effect of Different Concentration ofNitrogen and Magnesium Source

Assuming the reason for the stunned growth and

the yellowing of the leaves being deficiency in

minerals. Study were conducted on effect of

different concentration of nitrogen and

magnesium, For nitrogen source the ammonium

nitrate (Himedia) was added in different

concentration i.e. 10g/l, 15g/l and 20g/l in MS

medium (Himedia) with 4% sucrose

concentration and for magnesium sulphate

(Himedia) 10mg/l, 15mg/l and 20 mg/l was added

as the source of magnesium. On addition of these

nutrients to the media the better growth were

observed in the MS media containing shoot tip

explants with 4 % sucrose, 10 g/l of ammonium

nitrate and 10 mg/l of Magnesium sulphate as

extra addition to the MS media.

For multiplication of shoot induction, the MS

medium was supplemented with different

concentration of growth regulators, cytokinins 6-

Benzylaminopurine (BAP: 1, 1.5, 2, 2.5, 3, 3.5, 4,

4.5 and 5.0 mg /l), Kinetin (Kn: 1, 1.5, 2, 2.5, 3,

3.5, 4, 4.5 and 5.0 mg /l) and auxin 0.5 mg/l NAA

(Napthelic Acetic Acid) either individually or in

combination of cytokinin and auxin. The medium

was supplemented with 0.7 % agar (Himedia) the

pH of the medium was adjusted to 5.9 before

autoclaving at 121oC for 15 minutes. All the

cultured tubes were incubated under a 16 hr/ 8hr

light and dark photoperiod at 25 ± 2oC light

intensity in the culture room. Each treatment was

represented by 10 cultures and all the

experiments were carried out for three replicates.

After six weeks of inoculation, both shoot and root

was found in the same medium with multiple

shoots. The multiple shoots were subculture in

the same medium for 18 months and after 50

days of subculture plantlets were taken for

acclimatization for hardening process. Data were

scored based on visual observation and length

of shoot and root was measured with graded

measuring scale.

Acclimatization

The in vitro healthy rooted large cardamom

propagules were deflasked, further plantlets were

thoroughly washed under the running tap water

to remove all the traces of media from the

propogules, and treated with fungicides (Bavistin

2%) for 10-15 minutes.

The treated plantlets were then taken for

acclimatization to pot by placing transparent

polythene over pot for two weeks for maintaining

humidity. Different media/substrates were used

for acclimatization of plantlets. (A) Pot containing

a mixture of coco peat, soil, perlite and sand with

(1:1:2:1) ratio, (B) soil, cow dung and sand with

(1:1:1) ratio and (C) cow dung, soil, perlite and

sand with (1:1:1:1) ratio. Later the polythene cover

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was removed and directly transferred to fields for

secondary hardening.

STATISTICAL ANALYSIS

The statistical analysis with one-way analysis of

variance (ANOVA) using SPSS 16.0 analysis

software was executed to determine the

significance of differences between the

treatments (P<0.05) treated with different

concentration and combination of the growth

hormone on shoot number, root number and leaf

number. No significance differences were

observed (Table 1).

RESULTS AND DISCUSSION

The shoot tip as explants showed good response

while the leaf portion showed no growth response

was observed, the present result was

accordance with (Sajina et al., 1997) who reported

in vitro micro propagation large cardomom using

shoot tip explants. Among the various medium

used namely B5 (Gamborg media), full and 1/2

strength of MS media and WPM media. The

growth was observed only in MS media fortified

with different growth hormone concentration and

combination. Similar result of in vitro micro

propagation of large cardomom MS medium was

reported by (Sajina et al, 1997). The initiated

shoots showed stunned growth and yellowing of

leaves (Figure 4). The addition of 10 mg/l of

Table 1: ANOVA of Shoot Number, Root Number and Leaf Numberof Ammomum subalatum, The Analysis Showed No Significance

with Different Combination of Growth Regulator Treatments

Sample Source of Variation SS df MS F P-value

Shoot number Between Groups 52.27599 3 17.42533 0.762536 0.520662

Root number Between Groups 8.16666 3 8.16666 0.735340 0.400405

Leaf number Between Groups 8.813329 3 2.937776 0.540344 0.656971

Ammonium Nitrate and 10 mg/l of Magnesium

Sulphate with 4 % of sucrose showed good

numbers of shoots and roots growth in the media

(Figure 5). The shoots proliferation, mean

number of shoots, leaf, and roots and over a

period of 8 weeks were recorded in various

combinations of growth regulators (Tables 2, 3, 4

and 5). Shoot tip explants did not show shoot

initiation in the absence of growth regulators and

at the concentration of 4-5mg/l BAP and 4-5mg/l

Kinetin even after maintaining the cultures beyond

the normal observation period.

The organogenetic response of the vegetative

shoot tip buds of large cardomom is depicted in

(Figures 1 to 2). The maximum number of shoots

per explant was obtained for medium containing

BAP 3 mg/l and NAA 0.5 mg/l with 4 % sucrose,

followed by BAP 2.75 mg/l and NAA 0.5 mg/l with

4 % sucrose which were higher than all other 52

combinations (Figure 5). In this experiment,

Ramsay cultivar showed considerably good

response in the culture medium having

combination of BAP 3 mg/l and NAA 0.5 mg/l with

4 % sucrose l (Figure 5). The highest number of

shoots on the best combination of BAP 3 mg/l

and NAA 0.5 mg/l with 4 % sucrose ranged from

18-19 numbers of shoots per explants was

recorded with an average of 10 numbers of shoots

after incubation for 8 weeks of incubation (Table 2)

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Table 2: Effect of Growth Regulators (BAP and NAA) on Shoot Number, Root Numberand Leaf Number of Shoot Bud Explants of Ammomum subalatum

BAP mg/l NAA mg/l Shoot No. Root No. Leaf No.

1 0.5 0.00 0.00 0.00

1.25 0.5 0.00 0.00 0.00

1.5 0.5 1.50 3.00 4.21

1.75 0.5 1.25 3.00 4.78

2 0.5 2.03 2.00 6.32

2.25 0.5 10.05 5.00 4.53

2.5 0.5 18.03 6.00 6.87

2.75 0.5 15.06 5.00 5.43

3 0.5 19.02 4.00 8.47

3.5 0.5 3.00 3.00 4.00

4 0.5 0.00 0.00 0.00

4.5 0.5 0.00 0.00 0.00

5 0.5 0.00 0.00 0.00

Table 3: Effect of Growth Regulators (BAP) on Shoot Number, Root Numberand Leaf Number of Shoot Bud Explants of Ammomum subalatum

BAP mg/l NAA mg/l Shoot No. Root No. Leaf No.

1 0 0.00 0.00 0.00

1.25 0 1.20 3.10 4.02

1.5 0 1.30 3.00 4.43

1.75 0 2.02 3.00 4.45

2 0 2.03 4.00 5.04

2.25 0 9.34 4.00 4.35

2.5 0 7.04 6.00 5.03

2.75 0 9.43 6.00 4.32

3 0 11.02 12.00 6.38

3.5 0 3.00 5.00 4.00

4 0 0.00 0.00 0.00

4.5 0 0.00 0.00 0.00

5 0 0.00 0.00 0.00

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Table 4: Effect of Growth Regulators (Kinetin) on Shoot Number, Root Numberand Leaf Number of Shoot Bud Explants of Ammomum subalatum

Kinetin mg/l NAA mg/l Shoot No. Root No. Leaf No.

1 0 0.00 0.00 0.00

1.25 0 1.00 0.00 0.00

1.5 0 1.02 3.00 3.00

1.75 0 1.34 4.00 3.00

2 0 2.23 6.00 3.00

2.25 0 5.43 11.00 5.00

2.5 0 5.68 13.00 6.00

2.75 0 8.65 4.00 6.00

3 0 9.34 8.00 5.00

3.5 0 3.03 6.00 6.00

4 0 0.00 0.00 0.00

4.5 0 0.00 0.00 0.00

5 0 0.00 0.00 0.00

Table 5: Effect of Growth Regulators (Kinetin and NAA) on Shoot Number, Root Numberand Leaf Number of Shoot Bud Explants of Ammomum subalatum

Kinetin mg/l NAA mg/l Shoot No. Root No. Leaf No.

1 0.5 0.00 0.00 0.00

1.25 0.5 0.00 0.00 0.00

1.5 0.5 1.02 3.00 3.00

1.75 0.5 1.34 5.00 3.00

2 0.5 4.23 6.00 5.00

2.25 0.5 7.43 9.00 6.00

2.5 0.5 6.68 9.00 5.00

2.75 0.5 7.65 9.05 5.00

3 0.5 11.34 13.00 6.00

3.5 0.5 5.03 7.00 6.00

4 0.5 0.00 0.00 0.00

4.5 0.5 0.00 0.00 0.00

5 0.5 0.00 0.00 0.00

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Figure 1-8: Different Stages of In Vitro Micropropagation of Ammomum subalatum.cv-Ramsay Through Merismetic bud Culture on Modified MS Medium Fortified

with BAP (3 mg/l) and NAA (0.5 mg/l). 1. Merismetic Bud Explant at Culture; 2 Initiation ofShoot Tip Bud After 15-20 Days; 3 Formation of Multiple Shoots After 35 Days; 4 Established

Subculture Plant on Same Medium; 5 Clusters of Multiple Shoots and Roots After 50 Days;6 Formation of Roots on Same Hormonal Concentration; 7 Acclimatized Plant Completed for

Transferred to Field; 8 Transferred Six Months Old Plants with Clump Multiple Shooting

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which was significantly higher than reported by

(Sajina et al,1997), who found 10-12 numbers of

shoots per explants in modified MS. (Kambaska

et al, 2009) also reported maximum number of

shoots was obtained on the medium containing

BAP 2 mg/L and NAA 0.5 mg/L (7.7 shoots per

explants in cultivars- Suprava and Suruchi) in

ginger.

The regenerated micro propagules produced

a good number of roots in the same hormonal

concentration, which is in accordance with the

reports published by (Sajina et al, 1997; Pradhan

et al, 2010) (Figures 4-6). The present protocol

reduced the in vitro rooting step and showed this

protocol is cost effective for rapid multiplication

of large cardomom.

The maximum numbers of leaf numbers was

recorded in medium containing BAP 3 mg/l and

NAA 0.5 mg/l with 4 % sucrose recorded 8.47 ±

0.3 numbers of leaves followed by BAP 2.5 mg/l

and NAA 0.5 mg/l with 4 % sucrose recorded 6.87

± 0.4 numbers of leaves per explant (Figure 5).

When a single growth hormone was used in

the in vitro culture MS medium the maximum

number of shoots was recorded (11.02 ±0.4) in

BAP 3 mg/l followed by (09.34 ±0.2) numbers of

shoots in Kinetin 3 mg/l with lower number of leaf

and roots as compared with other treatment

(Figure 3).

Comparatively lower response was recorded

when BAP and Kinetin was added alone in the

medium this shows that the combination of

cytokinins and auxin in the culture medium

enhanced the response in a number of species

in terms of shoot growth (Table III and V). Similar

result was concur with Spathiphyllum floribundam

when cultured on media with BA alone, a limited

1.5 shoots per culture was examined; while

addition of IAA produced an average number of

11.6 shoots per explants (Malamug et al, 1991)Similar observation was reported in Hovenia

dulcis nodal culture (Echeverrigaray et al, 1998).

Same result was significantly coordinated with

the work done by (Kambasaka et al, 2009) after

addition of auxin produced more numbers of

shoots. Therefore MS medium supplemented

with cytokinins and auxins was suitable for in vitro

micropropagation large cardomom. Few

researchers also reported on micropropagation

of zingiberaceous species like cardamom, ginger

and turmeric also consequences the same

(Pradhan et al, 2010; Nadgauda et al, 1978;

Balachandran, 1990).

A healthy plantlets having 5-6 cm long shoot

with a good number of roots were taken for the

acclimatization or hardening of plantlets, with the

combination (A) of perlite, soil and farmyard

manure with ratio of 1:1:1 showed 90%

regenerated response (survived) of in vitro

plantlets followed by combination (B) pasteurized

soil, farmyard manure, perlite sand at the ratio

1:1:1:1 100 % survived and last combination soil,

sand, and manure at the ratio 1:2:1 (C) showed

70% surviving response towards the

acclimatization of plants as shown in (Figure 7).

After primary and secondary hardening then

transferred to field and leaves numbers and shoot

numbers measured and compared with in vivo

Ammomum subalatum control plants. Field

observation showed that micro propagated plants

found more resistant against diseases and

showed rapid multiple shooting during hardening

and after transfer into field. The mean of 20 plants

studied, the length and breadth of leaves (38.43

± 2.12) cm and (9.76 ± 1.67) cm were recorded

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respectively, followed by mean numbers of

multiple shoots (30.34 ± 4.23) were recorded in

6 months period with (13.56 ± 4.12) cm height

were recorded (Figure 8).

CONCLUSION

In vitro micro propagation of Ammomum

subalatum as a best alternative method of

propagation has proven to be a good method for

rapid clonal mass propagation for good and

healthy high yielding plant with minimum disease

to meet the supply for the huge demand. With

the problem of the fungal and viral diseases

causing mass wipe out of the crop, this method

can prove to be one of the best solution for this

problem. Since large cardamom farming is the

major source of income for most of the farmers

in Sikkim. This best alternative method of

propagation can bring huge benefit for the farmer

society of Sikkim.

This investigation revealed that the shoot tip

as explants sample of Ramsay responded well

and it was found to be the best explants for the

production of disease free planting materials. We

tried to explore 52 different hormone

concentrations using MS medium. The shooting

and the rooting of the explants were better and

faster with more number of healthy shoots, leaves

and proper growth in case of the modified MS

media fortified with growth regulator on different

concentration. The best medium was modified

MS medium fortified with BAP 3 mg/l + NAA 0.5

mg/l and sucrose 40g and for the formation of

both multiple shoots and roots and successfully

100% acclimatized plantlets were transferred into

fields.

ACKNOWLEDGMENT

Authors are thankful to Member Secretary, Sikkim

State Council Science and Technology, Gangtok,

Sikkim for providing for laboratory facilities.

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